Since the technology breakthroughs in the artificial fertilization field, culturing crustaceans using mass seed production and feed formulation techniques have rapidly turned the crustacean culture industry into one of the world's most commercially important business. Among the variety of crustaceans, shrimp, particularly penaeid shrimp, has become number one cultivated crustacean in southeast Asia, especially in Taiwan.
In recent years, the penaeid shrimp of southeast Asia are severely threatened by viral infections. According to recent reports, four baculoviruses, namely, white spot syndrome virus (WSSV), Penaeus monodon baculovirus (MBV), baculoviral midgut gland necrosis (BMN), and baculovirus penaei (BP), have been identified as the contributing pathogens which cause the high mortality in penaeid shrimps in southeast Asia.
Viral diseases cannot be cured by therapeutic reagents. The best ways to control the diseases include prevention through early detection and development of vaccines. In either way, the understanding of the biological, biochemical, and serological characteristics of the viruses is fundamentally required, which in turn requires the industry to have the capacity of mass producing the viruses, preferably through an in vitro cell culture system. So far, due to the lack of susceptible penaeid shrimp cell culture system, research on penaeid viruses has been limited to histopathological and transmission electron microscopic observation only. Therefore, the development of a new culture system which can be susceptible to WSSV, MBV, BMN, and/or BP is desperately in demand in order to control the wide spread of viral diseases in penaeid shrimps.
In 1990, an established fish cell line, epithelioma papillosum cyprini (EPC) originating from the carp, was reported. This cell line was found to be susceptible to the shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV) (U.S. Pat. No. 5,236,840). The establishment of EPC leads to the subsequent findings of cures for IHHNV. Unfortunately, EPC is not susceptible to WSSV, MBV, BMN, or BP.
Since then, there have been several other attempts by researchers to extend the culture life span of penaeid shrimp primary cell cultures. Unfortunately, none has successfully passed the cells beyond the primary stage.
In 1995, two reports describing the development of an in vitro subculture system using cells from the oka organ of Penaeus monodon were published (See Hsu et al. (1995), Aquaculture, 136:43-55, and Hsu et al. (1995), Proc. Internatl. Symposium on Biotech. Appl. in Aquaculture, 10:161-170, which are herein incorporated by reference). These two reports were authored by the same inventor as in the present invention. The cells from the reported subculture system had been maintained for more than 90 passages. The reports also showed that after 60 passages, the morphology of these cells changed from suspended cells to monolayers, which served as an early indication of the establishment of a cell line. However, the reports not only did not provide information as to the genetic and biochemical characteristics of the cell line, but also were silent on the susceptibility or mass production capability of the cell line to virus, especially shrimp virus and other fish or shellfish virus.
In the invention to be presented in the following sections, an establishment of an immortal cell line (PMO) will be introduced. This immortal cell line is developed from the cells described in the previous reports (id.) by passing the cells to more than 150 passages. Experimental results which show that the PMO cell line is susceptible to various shrimp, fish, and shellfish viruses, and has the capability of mass producing the viruses will also be described. The mass production of viruses using this PMO cell line provides resources for future studies on viral gene functions, virus-host cell interactions, and signal transductions after viral infection. Finally, because the PMO cells are derived from lymphoid organ, they may provide an experimental model for the future studies of the cellular and humoral immunity of shrimp.